Determinants of bacterial biofilm formation at the intestinal mucosal interface and their roles in pathogen exclusion

肠粘膜界面细菌生物膜形成的决定因素及其在病原体排除中的作用

• 批准号: 10612990
• 负责人: Josephine Ni
• 金额: $ 16.04万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10612990
• 关键词: Address; Anabolism; Animal Model; Bacteria; Bacterial Infections; Biologic Characteristic; Biological; Biological Assay; Biological Models; Body Weight decreased; Cell Adhesion; Characteristics; Chelating Agents; Chemicals; Citrobacter rodentium; Colitis; Colon; Colonic Diseases; Colorectal Cancer; Communities; Complement; Complex; Confocal Microscopy; Data; Dental caries; Dietary Iron; Disease; Engineered Gene; Engineering; Enterobactin; Environment; Escherichia coli; Exclusion; Genes; Goals; Growth; Harvest; Image; Immunofluorescence Immunologic; In Vitro; Inductively Coupled Plasma Mass Spectrometry; Infection; Infectious colitis; Inflammatory Bowel Diseases; Intestinal Diseases; Intestinal Mucosa; Intestines; Investigation; Iron; Knowledge; Length; Link; Microbial Biofilms; Microbiology; Modeling; Mucins; Mucous Membrane; Mucous body substance; Mus; Mutation; Nitrogen Fixation Genes; Nutrient; Pathogenesis; Pathway interactions; Pattern; Pennsylvania; Pentetic Acid; Physiological; Pilum; Play; Proteins; Proteomics; Radial; Research; Research Personnel; Role; Small Intestines; Specificity; Surface; Techniques; Testing; Universities; Urinary tract; career development; experience; experimental study; fitness; gain of function; gut colonization; gut microbiome; in vivo; inhibitor; innovation; iron supplementation; knowledge base; loss of function; microbial; microbiome analysis; microbiota; mucosal microbiota; murine colitis; mutant; pathogen; rRNA Genes; skills

Project Summary It is well established that biofilms are critical for the pathogenesis of many bacterial infections. More recently, biofilms have also been implicated in the pathogenesis of intestinal diseases such as colorectal cancer and inflammatory bowel disease, where mucosal-associated polymicrobial communities harvested from diseased colon exacerbates disease in animal models. Despite these studies, the presence of biofilms in the intestinal tract has been controversial. Since the mucus that lines the intestinal tract is constantly shed, the ability of this layer to provide a stable surface for biofilm formation has been questioned. There are currently no tractable models to understand or explore biofilm formation in the intestinal environment. As a result, evidence supporting a role for biofilm formation in colonization of the mucosal surface is lacking. In preliminary data, we show that deletion of the E. coli gene ntrC (∆ntrC), which encodes a regulator for nitrogen utilization, results in increased biofilm formation in vitro and confers a 2-3 log competitive advantage over wild-type (WT) E. coli in vivo in mice (despite reduced growth rate in vitro). These results are consistent with a role for biofilm formation in intestinal colonization. I therefore hypothesize that competitive fitness of ∆ntrC E. coli is a reflection of biofilm formation at the mucosal surface of the intestinal tract. The objective is to further characterize the role of biofilm formation in E. coli colonization of the intestinal tract and to use this model system to understand more generally the role of biofilms in disease pathogenesis at the intestinal mucosal interface. This hypothesis will be tested through three inter-related specific aims that will determine the relationship behind biofilm formation in vitro and spatially-localized colonization throughout the murine small intestine and colon (Aim 1), determine the role of iron in E. coli biofilm formation and intestinal colonization (Aim 2), and characterize the biological impact of bacterial biofilm formation in disease pathogenesis using a murine model of colitis (Aim 3). The experiments will use various innovative approaches including the creation and inoculation of genetically- modified E. coli strains, quantification of iron via ICP-MS in the intestinal mucus and lumen, and 16S rRNA gene microbiota sequencing to address these physiologically-relevant mechanisms. The proposed research is significant because it will provide new information about the bacterial determinants of biofilm formation in the intestinal tract, with the rationale of using them for competitive niche exclusion in models of colitis. University of Pennsylvania provides the perfect research environment to conduct this investigation given local expertise in the inter-related fields of gut microbiome and basic microbiology. The candidate will gain experience in compositional and functional analysis of the microbiome, acquire fundamental skills in microbial gene engineering techniques, and broaden her knowledge base of microbiology. These skill sets will greatly enhance the candidate's career development into an independent scientific investigator, with the long-term goal of enabling us to better understand and modulate the effects of the gut microbiome in disease states.

项目摘要 众所周知，生物膜对于许多细菌感染的发病机理至关重要。最近， 在肠道疾病（例如结直肠癌和 炎症性肠病，在该疾病中与粘膜相关的多数菌群从解剖中收获 结肠加剧动物模型中的疾病。尽管进行了这些研究，但在肠道中存在生物膜 道一直有争议。由于将肠道的粘液不断脱落，所以这种能力 质疑为生物膜形成提供稳定表面的层已受到质疑。目前没有可行的 在肠道环境中了解或探索生物膜形成的模型。结果，证据 缺乏支持生物膜形成在粘膜表面定植中的作用。在初步数据中，我们 表明大肠杆菌基因NTRC（∆NTRC）的删除编码用于氮利用的调节剂，导致 在体外的生物膜形成增加，并赋予野生型（WT）大肠杆菌的2-3对数竞争优势 小鼠体内（尽管体外生长速度降低）。这些结果与生物膜形成的作用一致 在肠道定植中。因此，我假设∆ntrc大肠杆菌的竞争适应性是生物膜的反映 肠道粘膜表面的形成。目的是进一步表征 肠道大肠杆菌定植的生物膜形成，并使用此模型系统了解更多 通常，生物膜在肠道粘膜界面上疾病发病机理中的作用。这个假设将会 通过三个相互关联的特定目的进行测试，这些目标将决定生物膜形成背后的关系 整个小肠和结肠的体外和空间定位的定植（AIM 1），确定 铁在大肠杆菌生物膜形成和肠道定植中的作用（AIM 2），并表征生物学 细菌生物膜形成在疾病发病机理中的影响（AIM 3）。这 实验将使用各种创新方法，包括创建和接种一般的 - 改良的大肠杆菌菌株，通过ICP-MS定量肠道粘液和腔内的铁和16S rRNA 基因微生物群测序以解决这些与物理相关的机制。拟议的研究是 重要的是因为它将提供有关细菌确定生物膜形成的新信息 肠道的理由是将它们用于结肠炎模型中的竞争利基排除。大学 宾夕法尼亚州提供了完美的研究环境，可以在本地专业知识中进行这项投资 肠道微生物组和基本微生物学的相互关联领域。候选人将获得经验 微生物组的组成和功能分析，获得微生物基因的基本技能 工程技术，扩大了她的微生物学知识基础。这些技能将极大地 长期将候选人的职业发展提升为独立的科学研究者 使我们能够更好地理解和调节肠道微生物组对疾病状态的影响。